Molecular-Based Taxonomic Inferences of Some Spider Mite Species of the Genus Oligonychus Berlese (Acari, Prostigmata, Tetranychidae)

Simple Summary Spider mites belonging to the genus Oligonychus Berlese are serious pests of various fruits, ornamentals, agronomic crops, and trees. To effectively manage these pests, their accurate identification is crucial. Morphological-based identification of Oligonychus species is very challenging. The present study used molecular data to identify/confirm the species identity of some Oligonychus species, including various samples lacking male specimens. Moreover, phylogenetic analyses validated the morphological-based subdivision of the genus Oligonychus. The integrative taxonomic approaches are vital for accurately identifying closely related Oligonychus species. Abstract DNA barcoding technology using short DNA sequences has emerged as an efficient and reliable tool for identifying, confirming, and resolving closely related taxa. This study used ITS2-rDNA and mtCOI DNA sequences to confirm the identity of eight Oligonychus species, representing 68 spider mite samples, collected mainly from Saudi Arabia (SA) and some from Mexico, Pakistan, USA, and Yemen. The intraspecific nucleotide divergences of the studied Oligonychus species ranged from 0% to 1.2% for ITS2 and 0% to 2.9% for COI. However, the interspecific nucleotide divergences were distinctly higher than the intraspecific ones and ranged from 3.7% to 51.1% for ITS2 and 3.2% to 18.1% for COI. Furthermore, molecular data correctly confirmed the species identity of 42 Oligonychus samples lacking males, including a previously claimed sample of O. pratensis from SA. High genetic variations were detected in two Oligonychus species: O. afrasiaticus (McGregor) (nine ITS2 and three COI haplotypes) and O. tylus Baker and Pritchard (four ITS2 and two COI haplotypes). In addition, ITS2- and COI-based phylogenetic trees confirmed the subdivision of the genus Oligonychus. In conclusion, integrative taxonomic approaches are vital to resolve the closely related Oligonychus species, identify the samples lacking male specimens, and assess phylogenetic relationships within and among species.


Introduction
In the genus Oligonychus Berlese (Acari, Prostigmata, Tetranychidae), morphologicalbased differentiation among species always depends on the characterization of the key trait of male aedeagus [1][2][3]; however, in the subdivision of Oligonychus into subgenera and groups/subgroups, the characterization of both male and female is compulsory [3]. Indeed, exact species identification in Oligonychus is usually a challenge because of the minute differences in aedeagus; limited diagnostic traits in females; and the presence of various species complexes, e.g., pratensis complex and punicae complex [1][2][3][4][5][6][7]. Moreover, the key diagnostic trait of the aedeagus becomes unreliable if males are not mounted accurately in their lateral positions, or are either described briefly without illustrations or

Collection, Preservation, and Processing of Spider Mite Samples
In total, 68 spider mite samples were collected from wild and cultivated vegetation in different seasons and localities from Mexico, Pakistan, SA, USA, and Yemen (Table S1; Figure 1). Out of 68 samples, 61 were collected from 10 Saudi provinces: Asir, Baha, Eastern Province, Jouf, Jizan, Makkah, Madinah, Qassim, Riyadh, and Tabuk (Table S1; Figure 2). The Saudi mite samples were collected between 2018 and 2021, except for one sample (sample voucher number/SVN: 39; Table S1), which was collected in 2013 from SA, and was claimed as O. pratensis [23]. The remaining 7 Oligonychus samples were received from 4 other countries: 1 from Mexico (SVN: 53), 1 from USA (SVN: 40), 2 from Yemen (SVN: 49 and 50), and 3 from Pakistan (SVN: 51, 66, and 67) (Table S1; Figure 1). Almost 2/3 of the mite samples had only female specimens (Table S1). Each sample's collection details were recorded, e.g., sample voucher number (SVN), collection date, locality, host plant, GPS coordinates, collector name, etc. (Table S1). The voucher specimens of male/female representing each collected sample were deposited at the King Saud University Museum of Arthropods (Acarology section), Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University (KSU), Riyadh, SA.     [24,25] at our request. Mite samples having male and female individuals were morphologically re-examined at the acarology laboratory (KSU) using the phase contrast microscope (BX51, Olympus, Tokyo, Japan) and identified till the species level following the taxonomic literature of the genus Oligonychus [3,26,27]. However, those 42 samples containing only female specimens were only identified up to the level of the genus Oligonychus, following Bolland, Gutierrez, and Flechtmann [26].    The concentration of genomic DNA solutions was assessed by the NanoDrop TM One spectrophotometer (Thermofisher Scientific, Waltham, MA, USA). The extracted DNA samples were immediately stored at −20 • C after labelling with the appropriate field information.

Phylogenetic and Genetic Distances Analyses
Phylogenetic investigations were performed to assess the evolutionary relationships within and among various Oligonychus species using MEGA-X [29]. Phylogenetic trees were made using the neighbor-joining (NJ) method by the Tamura-Nei model [30]. The tree branch robustness was tested using the bootstrap analysis with 1000 replications [31]. Moreover, the pairwise p-distances (interspecific and intraspecific genetic divergence) were also calculated using MEGA-X.  Table S1). However, the samples containing only female specimens (Table S1) were recognized till the level of the genus Oligonychus.

Results
At the molecular level, out of the 68 samples, 65 ITS2 amplicons were successfully amplified and sequenced. The length of the cleaned ITS2 sequences without ITS2 primers ranged from 405 to 539 bp (Table S1). However, the length of the 16 selected COI sequences obtained during this study was 410 bp (Table S1). Based on the ITS2 and COI data, the taxonomic identity of all 68 spider mite samples was successfully confirmed, which  Table S1) that lacked male specimens was recognized as O. afrasiaticus ( Figure 3; Table S2).
Insects 2023, 14, x FOR PEER REVIEW 6 of 14 Figure 3. NJ phylogenetic tree based on 33 ITS2 sequences, representing different samples of seven Oligonychus species. Eutetranychus orientalis* was retrieved from GenBank and used as an outgroup taxon. A total of 30 sequences were obtained in the present study from different hosts and regions in Saudi Arabia and one from USA (** including 19 samples that have not been identified morphologically till species due to the absence of males, and *** one sample that was previously claimed as O. pratensis from Saudi Arabia) [23]. One sequence of O. punicae was retrieved from GenBank, previously reported from Saudi Arabia [7]. Numbers on tree branches are bootstrap values obtained from 1000 replicates. . NJ phylogenetic tree based on 10 COI sequences, representing different samples of six Oligonychus species. Eutetranychus orientalis* was retrieved from GenBank and used as an outgroup taxon. A total of eight sequences were obtained in the present study from different hosts and regions in Saudi Arabia, and one from USA (** including samples that have not been identified morphologically till species due to the absence of males). One sequence of O. punicae was retrieved from Gen-Bank, previously reported from Saudi Arabia [7]. Numbers on tree branches are bootstrap values obtained from 1000 replicates. . NJ phylogenetic tree based on 33 ITS2 sequences, representing different samples of seven Oligonychus species. Eutetranychus orientalis* was retrieved from GenBank and used as an outgroup taxon. A total of 30 sequences were obtained in the present study from different hosts and regions in Saudi Arabia and one from USA (** including 19 samples that have not been identified morphologically till species due to the absence of males, and *** one sample that was previously claimed as O. pratensis from Saudi Arabia) [23]. One sequence of O. punicae was retrieved from GenBank, previously reported from Saudi Arabia [7]. Numbers on tree branches are bootstrap values obtained from 1000 replicates.
Insects 2023, 14, x FOR PEER REVIEW 6 of 14 Figure 3. NJ phylogenetic tree based on 33 ITS2 sequences, representing different samples of seven Oligonychus species. Eutetranychus orientalis* was retrieved from GenBank and used as an outgroup taxon. A total of 30 sequences were obtained in the present study from different hosts and regions in Saudi Arabia and one from USA (** including 19 samples that have not been identified morphologically till species due to the absence of males, and *** one sample that was previously claimed as O. pratensis from Saudi Arabia) [23]. One sequence of O. punicae was retrieved from GenBank, previously reported from Saudi Arabia [7]. Numbers on tree branches are bootstrap values obtained from 1000 replicates. . NJ phylogenetic tree based on 10 COI sequences, representing different samples of six Oligonychus species. Eutetranychus orientalis* was retrieved from GenBank and used as an outgroup taxon. A total of eight sequences were obtained in the present study from different hosts and regions in Saudi Arabia, and one from USA (** including samples that have not been identified morphologically till species due to the absence of males). One sequence of O. punicae was retrieved from Gen-Bank, previously reported from Saudi Arabia [7]. Numbers on tree branches are bootstrap values obtained from 1000 replicates. . NJ phylogenetic tree based on 10 COI sequences, representing different samples of six Oligonychus species. Eutetranychus orientalis* was retrieved from GenBank and used as an outgroup taxon. A total of eight sequences were obtained in the present study from different hosts and regions in Saudi Arabia, and one from USA (** including samples that have not been identified morphologically till species due to the absence of males). One sequence of O. punicae was retrieved from GenBank, previously reported from Saudi Arabia [7]. Numbers on tree branches are bootstrap values obtained from 1000 replicates.  Moreover, the Oligonychus species of the subgenus Reckiella and species of the subgenus Oligonychus clustered separately into two monophyletic clades with 100% and 93% bootstrap values, respectively (Figure 7). In addition, the subgenus Oligonychus clade (supported with 93% bootstrap value) is further divided into two sub-clades, the b-i subclade, representing the peruvianus species group (female with either eight or nine tactile setae on tibia I), and the b-ii sub-clade representing the coffeae species group (female with either five, six, or seven tactile setae on tibia I) (Figure 7). Similarly, Oligonychus species of the subgenus Reckiella (Figure 8, clade a), and species of the subgenus Oligonychus ( Figure  8, clade b) grouped separately into two monophyletic clades. The estimated pairwise p-distances for the ITS2 sequences showed that interspecific genetic divergence in various tested Oligonychus species ranged from 0.037 to 0.511 (3.7% to 51.1%; Tables S2 and S6), and intraspecific divergence ranged from 0.000 to 0.012 (0% to 1.2%; Table S4). The pairwise p-distances of COI sequences among various Oligonychus species showed interspecific divergence ranging from 0.032 to 0.181 (3.2% to 18.1%; Tables S3 and S7); whereas, the intraspecific divergence within same species ranged from 0.000 to 0.029 (0% to 2.9%; Table S5). Exceptionally, three COI sequences retrieved from the GenBank (AB683664 from Japan; GU329963 from China, and X80865 from France), representing three populations of O. ununguis, showed high intraspecific genetic divergence (6.5% to 11%;  Figure 3). Similarly, in the COI-based NJ tree, the clade of the subgenus Reckiella and the subgenus Oligonychus grouped separately with monophyletic clades (Figure 4).
Moreover, the Oligonychus species of the subgenus Reckiella and species of the subgenus Oligonychus clustered separately into two monophyletic clades with 100% and 93% bootstrap values, respectively (Figure 7). In addition, the subgenus Oligonychus clade (supported with 93% bootstrap value) is further divided into two sub-clades, the b-i sub-clade, representing the peruvianus species group (female with either eight or nine tactile setae on tibia I), and the b-ii sub-clade representing the coffeae species group (female with either five, six, or seven tactile setae on tibia I) (Figure 7). Similarly, Oligonychus species of the subgenus Reckiella (Figure 8, clade a), and species of the subgenus Oligonychus (Figure 8, clade b) grouped separately into two monophyletic clades. Figure 7. NJ phylogenetic tree based on 16 ITS2 sequences, representing 15 Oligonychus spe Eutetranychus orientalis* was used as an outgroup taxon. A total of 11 Oligonychus species belon the subgenus (a) Reckiella, whereas four Oligonychus species (** including a cryptic Oligonychu previously claimed as O. punicae from Mexico) [7] belong to the species groups (b-i) peruvianus (b-ii) coffeae of the subgenus Oligonychus [3]. Numbers on tree branches are bootstrap value tained from 1000 replicates. Figure 8. NJ phylogenetic tree based on 33 COI sequences, representing 32 Oligonychus species tetranychus orientalis* was used as an outgroup taxon. A total of 12 Oligonychus species belong t subgenus (a) Reckiella, whereas 18 Oligonychus species (** including a cryptic Oligonychus sp., p ously claimed as O. punicae from Mexico) [7] belong to the species groups (b-i) coffeae and peruvianus of the subgenus Oligonychus [3]. Numbers on tree branches are bootstrap values obta from 1000 replicates. Eutetranychus orientalis* was used as an outgroup taxon. A total of 12 Oligonychus species belong to the subgenus (a) Reckiella, whereas 18 Oligonychus species (** including a cryptic Oligonychus sp., previously claimed as O. punicae from Mexico) [7] belong to the species groups (b-i) coffeae and (b-ii) peruvianus of the subgenus Oligonychus [3]. Numbers on tree branches are bootstrap values obtained from 1000 replicates.
The absence of male specimens can lead to the creation of doubtful Oligonychus species, especially in the absence of integrative taxonomic approaches [3]. For example, O. changi (Tseng), O. jiangxiensis (Ma and Yuan), O. longus (Chaudhri, Akbar, and Rasool), and O. pongami (Sivakumar and Kunchithapatham) are suggested as species inquirendae, which were described without males [3,11,[36][37][38]. In the present study, the molecular data revealed the correct species identity of 42 mite samples that lacked male specimens. Based on the low intraspecific nucleotide divergence (0% to 1.2%, ITS2; 0% to 2.9%, COI) and phylogenetic analyses, 16 [23], was correctly recognized as O. afrasiaticus. The absence of males in the Oligonychus population collected from grasses in the Baha region of SA led to the misidentification as O. pratensis [23]. Indeed, while conducting this study, we have not found any population of O. pratensis from SA. When we compared all the Saudi samples of Oligonychus belonging to the subgenus Reckiella with the Californian sample of O. pratensis collected from the USA (the country of its type locality), high genetic divergence either based on ITS2 (>11%) or COI (>8%), was shown, which is in accordance with separate species in previous molecular studies on tetranychid mites [7,14,16].
The wide distribution and polyphagous behavior of a spider mite species may affect its genetic structure [17,39]. The current study also revealed high genetic variations (number of haplotypes) in two widely distributed oligophagous species O. afrasiaticus and O. tylus collected/analyzed from India, Israel, Pakistan, SA, and Yemen. Nine ITS2 and three COI haplotypes have been identified in the date palm mite O. afrasiaticus. Out of the nine ITS2 haplotypes, representing seven hosts and four countries, six (H3, H4, H5, H6, H8, and H9) were only recovered from SA, one from both SA and Israel (H1), one from SA and Yemen (H2), and one (H7) from Pakistan. The geographic isolation may explain the distinction between the Middle Eastern and Pakistani ITS2 haplotypes of O. afrasiaticus [40]. Geographic isolation was a key factor in generating genetic variations among various populations of the citrus red mite Panonychus citri (McGregor) [41]. Similarly, O. tylus showed four ITS2 haplotypes representing nine localities and two COI haplotypes representing four localities, recovered from seven hosts and two countries. In addition to geographic distribution, host plants can increase genetic variations, as exhibited by the polyphagous spider mite species Eutetranychus orientalis (Klein) [17]. The oligophagous O. coniferarum inhabiting different species of conifers and the monophagous O. washingtoniae inhabiting W. filifera, did not show any genetic variations among their populations collected from different localities of SA.
The phylogenetic trees constructed using ITS2 sequences of 15 Oligonychus species and COI sequences of 30 Oligonychus species confirmed the morphological-based subdivision of the genus Oligonychus into two subgenera and four species groups [3]. The phylogenetic trees showed the monophyly nature of the two subgenera of Oligonychus: Reckiella and Oligonychus. These results are in agreement with previous findings either based on DNA-Sequence data of ITS2, COI, and 28S regions [14,16,32,42], or RNA-Sequence data [43], which separated Oligonychus species into two clades: a group of species with upturned (the subgenus Reckiella) and others with the downturned direction of male aedeagus (the subgenus Oligonychus). Further, in ITS2/COI-based phylogenetic trees, species belonging to the coffeae species group were clustered distantly from O. perseae of the peruvianus species group. It confirmed the morphological-based subdivision of the subgenus Oligonychus into two species groups based on a female character, i.e., the number of tactile setae on tibia I [3].
In addition, the molecular analysis of COI sequences of O. ununguis available on GenBank showed that the three cryptic species are hidden under the ununguis complex. The Japanese (Accession no: AB683664), Chinese (Accession no: GU329963), and French (Accession no: X80865) sequences of O. ununguis showed high genetic divergences (6.5% to 11%) from one another. Such high nucleotide divergences fall within the range of interspecific genetic divergence, as observed in the present (3.2% to 18.1%) and previous studies (5.4% to 18.3%) on Oligonychus species [7,14]. Similarly, the two GenBank ITS2 sequences of O. ununguis from China and Korea were previously highlighted as doubtful [7].
Some ITS2 (Accession no: MG429138, MG677944, OL830813, MK386956, India) and COI (KP180428, USA; MF462136, Australia) sequences available on GenBank identified to the genus Oligonychus level. Molecular data could not assign them to any of the known Oligonychus species. The high genetic divergences of ITS2 (4.4% to 10.4%) and COI (12.6%) clearly indicate their taxonomic identity as separate Oligonychus species. However, it is strongly recommended that the specimens/vouchers of these sequences need to be morphologically and molecularly re-characterized to know the actual global fauna of the genus Oligonychus.

Conclusions
In conclusion, the molecular data of ITS2 and COI successfully confirmed the taxonomic identity of 68 spider mite samples collected either with or without male specimens, representing eight Oligonychus species from Mexico, Pakistan, SA, USA, and Yemen. The phylogenetic analyses of various Oligonychus species also validated the subdivision of the genus Oligonychus into subgenera and species groups. The present study highlights the importance of integrated taxonomic approaches, e.g., the combination of morphological and molecular data, to confirm the identity of closely related Oligonychus species, especially in the absence of key morphological characters or individuals. Additionally, molecular data showed that the O. ununguis sequences available on the NCBI-GenBank database might be taxonomically misidentified and need further work. Overall, a taxonomic revision is compulsory to resolve the global issue of species complexes by adopting integrative taxonomic approaches.  Table S2: Genetic divergence (pairwise p-distance) based on 33 ITS2 partial sequences, either obtained in the present study or retrieved from GenBank, among seven Oligonychus species (Acari: Prostigmata: Tetranychidae), collected/analyzed from Saudi Arabia and the USA. Table S3: Genetic divergence (pairwise p-distance) based on nine COI partial sequences, either obtained in the present study or retrieved from GenBank, among six Oligonychus species (Acari: Prostigmata: Tetranychidae), collected/analyzed from Saudi Arabia and the USA. Table S4: Genetic divergence (pairwise p-distance) based on 38 ITS2 partial sequences, either obtained in the present study or retrieved from GenBank, showing variations among various populations of Oligonychus afrasiaticus and O. tylus (Acari: Prostigmata: Tetranychidae), collected/analyzed from Israel, India, Pakistan, Saudi Arabia, and Yemen. Table S5: Genetic divergence (pairwise p-distance) based on 12 COI partial sequences, either obtained in the present study or retrieved from GenBank, showing variations among various populations of Oligonychus afrasiaticus and O. tylus (Acari: Prostigmata: Tetranychidae), collected/analyzed from Israel, India, Saudi Arabia, and Yemen. Table S6: Genetic divergence (pairwise p-distance) based on ITS2 sequences among 15 Oligonychus species (Acari: Prostigmata: Tetranychidae), either obtained in the present study or retrieved from GenBank, analyzed from India, Mexico, Saudi Arabia, and the USA. Table S7: Genetic divergence (pairwise p-distance) based on COI sequences among 32 Oligonychus species (Acari: Prostigmata: Tetranychidae), either obtained in the present study or retrieved from GenBank. References [7,23] are cited in the Supplementary Materials.